1. Field of the Invention
This invention relates to an immunoassay of an analyte and materials used therein, and more particularly relates to a method and materials for immunoassay which does not require separation of bound and free fractions.
2. Description of the Prior Art
A variety of assay systems which are both rapid and sensitive has been developed to determine the concentration of a substance in a fluid. Immunoassays depend on the binding of an antigen or hapten to a specific antibody and have been particularly useful because they give high levels of specificity and sensitivity. These assays generally employ one of the above reagents in labeled form, the labeled reagent often being referred to as the tracer. Immunoassay procedures may be carried out in solution or on a solid support and may be either heterogeneous or homogeneous. Heterogeneous assays require a separation of bound tracer from free (unbound) tracer. Homogeneous assays do not require a separation step and thereby provide significant advantage in speed, convenience and ease of automation over heterogeneous assays.
Radioimmunoassay (RIA) procedures use radioisotopes as labels, provide high levels of sensitivity and reproducibility, and are amenable to automation for rapid processing of large numbers of samples. However, all RIA procedures require a separation step, since the parameter measured (nuclear decay) cannot be controlled by changing assay conditions or components. In addition, isotopes are costly, have relatively short shelf lives, require expensive and complex equipment, and extensive safety measures for their handling and disposal must be followed.
Enzymes have also been used as labels in immunoassay. Enzymeimmunoassay (EIA) may be homogeneous and does not require precautions against radioactivity. Conjugation of an enzyme with a protein is usually straightforward, and the resulting proteinenzyme conjugate is generally stable. However, EIA depends on the reaction of the enzyme conjugate with a substrate to produce a color which is measured, and thus requires the additional step of providing an enzyme substrate. In addition, sufficient time must be allowed for color development and an expensive spectrophotometer for measuring color change must be provided.
Some of the above disadvantages associated with RIA or EIA have been overcome by use of fluorochromes as labels in immunoassay. Fluoroimmunoassay (FIA) provides direct detection of the label and is readily adaptable to homogeneous assay procedures. However, known homogeneous FIA methods using organic fluorochromes, such as fluorescein or rhodamine derivatives, have not achieved the high sensitivity of RIA or EIA, largely because of light scattering by impurities suspended in the assay medium and by background fluorescence emission from other fluorescent materials present in the assay medium. Scattering is particularly troublesome with fluorochromes having a short (50 nm or less) Stroke's shift (the difference between the wavelength of the absorption and emmission). For example, the Stoke's shift of fluorescein isothiocyanate in only 20-30 nm. Background fluorescence is particularly troublesome when the assay medium is serum. The sensitivity of an assay in serum may be reduced up to one hundred fold compared to an identical assay in buffer.
The development of time-resolved fluoroimmunoassay (TR-FIA) has contributed to overcoming these problems. In this procedure, a fluorochrome label with a relatively long fluorescence emission decay time is excited with a pulse of light, and fluorescence emission from the label is measured after a preselected delay. Background emission of short decay time (generally less than 10 ns) essentially ceases during the delay and thereby does not interfere with measurement of the specific emission from the label. TR-FIA is most effective when the fluorescent label has a decay time of 100-1000 ns and a long Stoke's shift (100 nm or greater).
A class of labels meeting the requirements of TR-FIA is the lanthanide chelates. Lanthanide ions, in particular ions of europium and terbium form highly fluorescent chelates of long Stoke's shift (up to 250 nm) with organic ligands, in particular with .beta.-diketones. The ligand portion of the chelate absorbs excitation light and transfers the absorbed energy to the chelated metal ion. The metal ion emits the energy as fluorescence of exceptionally long decay time (1 ms). A discussion of the use of lanthanide chelates in TR-FIA is given in Analytical Biochemistry, 137 335 (1984).
U.S. Pat. No. 4,058,732 to Wieder discloses a method and apparatus for use of lanthanide chelates and time resolution in analytical fluorescent spectroscopy.
U.S. Pat. No. 4,283,382 to Frank et al. discloses an improvement in TR-FIA in which a lanthanide chelate label is incorporated into a polymeric bead lattice to eliminate water-induced quenching of the fluorescence emission of the label.
U.S. Pat. No. 4,374,120 to Soini et al. discloses increased stability of lanthanide chelates achieved by a 1:1:1 chelate of lanthanide, .beta.-diketone, and an aminopolycarboxylic acid analogue having a functional group useful for binding the chelate to a protein.
European patent application EP 0,064,484-A2 discloses a TR-FIA procedure in which the substance to be determined is coupled to an lanthanide by an aminocarboxylic acid analogue, and, after incubation, the lanthanide is split from the substance to be determined and chelated to a .beta.-diketone before detection.
Although the above methods have improved FIA, there is still a need for a FIA of high sensitivity which can be carried out rapidly without requiring a separation of bound and free fractions.